In its natural form, mammalian hemoglobin is tetrameric having a molecular weight of approximately 65 kD. Hemoglobin is structurally comprised of two alpha and two beta subunits with the alpha/beta subunits forming pairs. Each subunit contains a heme group and a polypeptide chain, called globin. In mammals, hemoglobin is encapsulated in erythrocytes (red blood cells), by a cell membrane which consists of proteins, phospholipids and cholesterol. See Clinical Hematology, by Wintrobe, 6th ed., pages 138-199, (1967).
Hemoglobin exists in equilibrium between the tetrameric and dimeric (approximately 32 kD) forms (Bunn, H. F. et al., Transperiod Assn. Am. Physicians, 81:187, (1968)). Equilibrium is influenced by concentration, oxygenation state, and temperature. When present extracelluarly, the dimers and tetramers are excreted by the kidney and are rapidly (half-life time 2 to 4 hours) removed from the circulatory system. Stabilized tetramers have a half-life of about 6 to 15 hours, while polymers remain in the blood stream longer. Typically, solutions comprising non-cross-linked hemoglobin have a circulating half life of 2-4 hrs when administered to an animal. Furthermore, unstabilized and even cross-linked tetrameric hemoglobin can be harmful to humans due to toxicity to the kidneys. Accordingly, polymers are the preferred form. To increase the half-life of hemoglobin in blood circulation and to reduce potential harmful effects of lower molecular weight forms of hemoglobin, native hemoglobin has been cross linked using a cross-linking agent to produce a stabilized form, so that dimer levels are significantly reduced. Polymers of hemoglobin with molecular weight greater than about 65 kD have been formed using polymerizing agents. In some cases, the cross-linking and the polymerizing agent are the same.
For use in humans, blood-substitutes comprising hemoglobin generally should include no more than 10% unstabilized hemoglobin. Unfortunately, polymerization of native hemoglobin by cross-linking can result in a solution wherein 50% or more of the hemoglobin is unstabilized or stabilized tetrameric hemoglobin, which generally is an unacceptably high level of these forms of hemoglobin. Therefore, a blood-substitute suitable for human use generally requires the removal of at least a portion of unstabilized or stabilized tetrameric hemoglobin to acceptable levels, resulting in significant waste of the starting material and high production costs.
For example, hemoglobin polymerized by exposure to a polymerizing agent is typically passed through a 100 kD molecular weight filter to remove the lower molecular weight hemoglobin from hemoglobin polymers. The lower molecular weight hemoglobin passes through the filter and into the filtrate. The filtrate is discarded as waste, typically resulting in a loss of up to about 50% of the isolated hemoglobin.
Therefore, a need exists to improve the efficiency of the hemoglobin polymerization process to improve the yield of polymerized hemoglobin blood-substitute from isolated hemoglobin.